16961PCMDRML FLUOR_ARTIFACT DRRml C and D fluorescent artifact dose-response confirmationMolecular Library Screening Center Network (MLSCN)Penn Center for Molecular Discovery (PCMD)Assay Provider: Michael McNeil, Colorado State University, Fort Collins, COMLSCN Grant: DA024889-01This screen is for compounds that have the potential to be developed into new drugs against tuberculosis (TB) because they inhibit the enzymes required for the formation of the cell wall of the tuberculosis bacterium. New drugs are needed because the rate of cure with the present drugs is very slow, and prevalence of Mycobacterium tuberculosis resistance to present drugs is increasing. Recently, an increase in co-infection of HIV and M. tuberculosis has occurred, and treatment with present drugs results in harmful HIV/TB drug interactions. To identify potential anti-TB agents, we focused on two enzymes that act sequentially in the formation of dTDP-rhamnose (dTDP-Rha), a biosynthetic precursor required for TB cell wall formation and found to be essential for the growth of M. smegmatis and M. tuberculosis. Active compounds inhibit the activity of the two enzymes, dTDP-6-deoxy-D-xylo-4-hexulose 3,5-epimerase (RmlC) and dTDP-6-deoxy-L-lyxo-4-hexulose reductase (RmlD). Enzyme activity is measured by the decrease in fluorescence upon the oxidation of NADPH to NADP. The enzymes are balanced to allow detection of an inhibitor of either enzyme. We have earlier reported the results of screening 200,000 compounds of the MLSCN library (AID 1532 and AID 1533), and tested compounds that gave >30.0% inhibition in dose-response (AID). Compounds giving IC50 <55 uM were tested in the dose-response assays in the absence of the Rml C and Rml D enzymes to rule out fluorescent artifacts. Compounds giving a dose-response in the absence of enzyme were judged to be fluorescent artifacts and excluded for further study.MATERIALSMOPS, Triton X-100 and TDP-Glc were purchased from Sigma. MgCl2 and glycerol were purchased from Fisher.Assay plate---Black 384-well plate (Corning 3676)Serial dilution compound plate---Polypropylene 384-well V-bottom plate (Greiner 781280)ASSAYThe screen for inhibitors of the Mycobacterium tuberculosis cell wall enzymes, RmlC and RmlD, is based on the decrease in fluorescence observed upon the oxidation of NADPH to NADP. Change in fluorescence is calculated from the difference between the fluorescence read at the beginning of the assay and one after 90 min. Compounds were tested in the absence of enzyme and substrates to determine if they affected the change in fluorescence over the 90 min timecourse. DOSE-RESPONSE PROTOCOL: 1. Serial dilute each compound in DMSO, 16 two-fold dilutions from 2.5 mM to 76 nM, using tips on Evolution. 2. Dispense 4 ul of water into 384-well assay plate using Multidrop.3. Pintool transfer compound twice (2x110 nL) from a 384-well dilution dose-response plate into a 384-well assay plate. 4. Add 5 ul 83.5 mM MOPS buffer. Final buffer composition: 50mM MOPS, pH 7.4 with 1 mM MgCl2, 10% glycerol and 0.01% TritonX-100. 5. Read NADPH fluorescence (Excitation 340/Emission 460 nm) at time 0 and after 90 min, at RT.DATA ANALYSISDose-response curves were plotted in Excel. Each compound column (3-22) contained 16 two-fold dilutions of a single compound, ranging in concentration from 55 #M to 1.7 nM. Percent activity was calculated for each concentration of each compound from the change in fluorescence over 90 min (&#8710;Signal), calculated from t=0 and t=90 min reads, and the mean of the change in plate controls and blanks over 90 min, using the following equation:% Activity = 100*((&#8710;signal-&#8710;blank mean)/(&#8710;control mean-&#8710;blank mean))ACTIVITY SCORINGNo dose-response = 0Dose-response = 40ACTIVITY OUTCOMENo dose-response = inactiveDose-response = activeANALYSIS OF SCREENING RESULTSCompounds that gave IC50s in the previous Rml C & D dose-response confirmation were tested in buffer alone, in the same IC50 format, to determine if they were false positives. Most of the compounds tested for IC50 gave IC50 curves independent of enzymes indicating that they are false positives. Of the compounds tested, only 14 gave no dose-response in the absence of enzymes; these are labeled as inactive is this assay and have been selected for further study.All of the compounds giving a dose-response in the absence of enzyme are fluorescent at 340/460 nm. It appears that these compounds show a small increase in fluorescence over the 90 min timecourse; because enzyme activity is measured by a decrease in fluorescence over 90 min, these compounds appear to be inhibitors. The effect is dependent on compound dose; the change as a fraction of total fluorescence remains constant, so the absolute increase in fluorescence increases with compound concentration.CONTRIBUTORSThis assay was submitted to the PCMD (Scott L. Diamond, Director; University of Pennsylvania) by Michael McNeil (PI) Colorado State University, Fort Collins, CO. Assay development and HTS was carried out by Sharmila Sivendran and data were submitted by Sharmila Sivendran and Andrew Napper.CORRESPONDENCEPlease direct correspondence to Sharmila Sivendran (ssharm@seas.upenn.edu).15321533887352RmlC888704rmlD dTDP-6-deoxy-L-lyxo-4-hexulose reductase RmlD83332M. tuberculosishttp://www.seas.upenn.edu/~pcmdPenn Center for Molecular Discovery1Dose-response41dTDP-6-deoxy-L-lyxo-4-hexulose reductase RmlD [Mycobacterium tuberculosis H37Rv]156104021Mycobacterium tuberculosis H37Rvtaxon83332RmlDdTDP-4-dehydrorhamnose 3,5-epimerase RmlC [Mycobacterium tuberculosis H37Rv]156106011Mycobacterium tuberculosis 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